The deaths by colon cancer are increasing. The number of deaths by colon cancer is the fourth largest among male, and the second largest among female deaths in all cancer deaths (Statistics of Japanese cancer deaths in 1999). According to an estimation of cancer patients in 2015 in Japan, number of colon cancer patients is estimated to be the first in both male and female. Global measures to counter colon cancer including secondary prevention are thus required, and mass screening of cancer may be one of the most effective methods.
For the mass screening of cancer, it is important that the detection method is easy and non-invasive. The only non-invasive method now available is the method to examine existence of occult blood in feces, that is, the fecal occult blood test, and is used extensively as a standard method of the mass screening of colon cancer.
However, the fecal occult blood test has rather low sensitivity and specificity (the sensitivity: 30 to 90%, the specificity: 70 to 98%), because appearance of hemoglobin in feces is not specific to tumor. Therefore, there is a shortcoming that quite a few false negatives and false positives exist.
Also, in the diagnosis of colon cancer, after or in parallel with the screening by the immunological fecal occult blood test, total colonoscopy or a combination of Ba-enema and sigmoidoscopy has been adopted. There is thus a shortcoming that it needs much time and effort.
As alternative methods to the fecal occult blood test, methods using DNA are reported, such as detection of mutations in K-ras, p-53, or APC genes, or detection of microsatellite instability in feces (D. Sidransky, et al., Science, 256, Apr. 3, 1992, 102-105; S. M. Dong, et al., Journal of the National Cancer Institute, 93 (11), Jun. 11, 2001, 858-865; G. Traverso, et al., The New England Journal of Medicine, 346 (5), Jan. 31, 2002, 311-320; G. Traverso, et al., The Lancet, 359, Feb. 2, 2002, 403-404).
These methods using DNA are non-invasive and can capture the direct changes in cancer cells, and have characteristics of having high specificity, and so are considered to be a hopeful method in the future. However, it has a demerit that the sensitivity is lower compared to the fecal occult blood test, a prior art, and is rather time and effort-consuming.
Further, as an alternative method to the fecal occult blood test, in order to detect gene expression more directly, a method for detecting mRNA of protein kinase C (PKC) or the like in the feces has been developed (L. A. Davidson, et al., Carcinogenesis, 19(2), 1998, 253-257; R. J. Alexander and R. F. Raicht, Digestive Diseases and Sciences, 43(12), 1998, 2652-2658; T. Yamao, et al., Gastroenterology, 114(6), 1998, 1198-1205).
However, the method making use of RNA described above could not have the sensitivity exceeding that of the fecal occult blood test method, because it was impossible to extract RNA easily and efficiently from a small amount of feces.
A method to detect RNA qualitatively and quantitatively by combining the PCR method with the reverse transcriptase reaction (RT), has been known. This RT-PCR method is superior to Northern blot technique in the high sensitivity to be able to detect trace molecules, and is more advantageous than the in situ hybridization technique in speed and easiness of manipulation.
However, since RNA is more unstable compared with DNA and is always subjected to a danger of decomposition by RNA digesting enzymes (RNases) which are ubiquitous in all the biological samples and very stable, strict control to avoid contamination of RNases is necessary in the RT-PCR method, during and after purification processes of RNA.
Therefore, when RNA is extracted from the feces, which is a biologically very crude sample, a process to separate the cell fraction in advance has been necessary, to exclude effects of RNases.
Accordingly, it has been considered impossible to extract RNA directly from feces containing enormous amount of RNases derived from very large amount of microorganisms, and, a separation of the cell fraction has been considered to be essential for removing at least exogenous RNases derived from microorganisms or the like.
Surprisingly, however, the inventor of the present invention found that in some cases, homogenization of frozen biological materials in the presence of RNase inhibitors can resolve the problems described above, and has completed the present invention.